FSH is a pituitary heterodimeric glycoprotein hormone synthesized and released from gonadotrope cells of the anterior pituitary gland. As a circulating hormone, FSH interacts with high affinity with receptor molecules on the surface of granulosa cells in the ovary. This interaction evokes a series of intracellular events, including a cyclic AMP second message and elicitation of a steroidogenic response by the granulosa cells resulting in estrogen production. Local estrogen and FSH stimulation promote the growth and maturation of ovarian follicles.
The amount of circulating FSH is dependent upon several other endocrine and neural factors. Gonadotropin releasing hormone (GnRH) is a peptide elaborated by neurons of hypothalamus. Released GnRH interacts with receptors on pituitary gonadotrope cells to control the synthesis and release of FSH and LH from the anterior pituitary gland. FSH secretion is also effected by circulating levels of steroid hormones. The steroidogenic response of granulosa cells to FSH results in gradually increasing estradiol levels. When serum estradiol reaches a critical level, it triggers a large increase in the rate of LH and FSH release from the anterior pituitary. The resultant LH surge induces ovulation and luteinization of granulosa cells. Progesterone is released from the corpus luteum following ovulation and this steroid prepares the uterus for implantation of the fertilized ovum. Elevated levels of estrogens and progesterone exert a negative feedback inhibition at hypothalamic sites to lower FSH and LH synthesis and release. Hence, the effects of steroids on gonadotropin release depend on the circulating levels; at low levels of estrogen, FSH and LH are positively regulated while higher levels result in negative feedback inhibition.
In males, FSH induces spermatogenesis through a proliferative effect on spermatocytes. Sperm production also requires testosterone, which is under positive regulatory control by LH.
An apparent paradox of the above-described hormonal control process is the production of both LH and FSH by the gonadotrope cell while GnRH serves as a positive regulatory agent for both hormones. Work within the last 10 years suggests that the peptides, activin, follistatin and inhibin selectively regulate FSH secretion from the anterior pituitary gland. FSH synthesis and release is activated by activin while inhibin and follistatin have negative feedback effects. The inhibitory effect of follistatin is thought to be mediated by its high-affinity binding to activin and blockage of its biological activity. There is evidence for both autocrine and paracrine local regulatory effects of these peptides and for feedback effects of inhibin released from gonadal tissue. Both inhibin and activin are structurally related and are members of the diverse transforming growth factor beta family of peptides. Study of the physiological roles of activin, follistatin and inhibin is a current area of active research (Reviewed by Knight, 1996).
FSH has been used extensively as a drug to treat human infertility by induction of follicular development in females. Earlier products were crude preparations of LH and FSH, i.e., Pergonal.RTM.. Products that are more recent have been purer FSH preparations. Metrodin.RTM., has low levels of LH and the FSH specific activity is about 100 IU/Mg. This drug requires intramuscular injections every day for 5 to 7 days, followed by a single injection of hCG to induce ovulation. A recent advance is Fertinex.RTM. which is affinity pure FSH, purified from hMG. This product exhibits a potency of 8500 to 13,500 IU/Mg at 95% purity. The high purity of Fertinex.RTM. allows delivery by subcutaneous injection, which can be administered at home. Following administration of Fertinex.RTM., hCG is used for induction of ovulation. Depending on the dosage of FSH administered, it may be used to promote in vivo fertility or, at higher dosages, it may be used to induce multiple oocyte formation for in vitro fertilization procedures. Recombinant forms of FSH (Puregon.RTM. and Gonal.RTM.) also are used as fertility drugs.
FSH has been purified from pituitary glands, human postmenopausal urine and from culture media collected from genetically engineered cells. FSH purification has been an active area of research over the past 30 years. Older methods rely on procedures such as ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis and chromatography on hydroxylapatite. In the method of Roos (1968) a FSH preparation of 14,000 IUs biological activity/Mg was obtained from fresh frozen human pituitary glands with an overall recovery of activity of 5.0%. A similar procedure applied to urinary FSH resulted in a preparation of 780 IU/Mg at a 7.7% overall yield. Because of the similar physicochemical properties of FSH and LH, i.e., similar molecular weight and overlapping isoelectric profiles, affinity chromatography methods have been employed to improve the separation of LH and FSH. In addition, affinity methods afford the possibility of high purification in a single step (up to 100-fold) thereby reducing the number of steps in a purification method and improving overall yield. The latter is a critical factor in the commercial production of FSH as overall yield is a major determinant of cost. Group specific affinity adsorbents such as the lectin Concanavalin A or chitosan (Japanese patent number 8,027,181) bind glycoproteins via specific carbohydrate groups. While these ligands are ineffective in the separation of two glycoproteins such as FSH and LH, Concanavalin A has been used to characterize microheterogeneity of purified FSH preparations (e.g., Chappel, et.al., 1983).
Immunoaffinity chromatography (IAC) relies upon the specificity of mono- or polyclonal antibodies for capture of specific protein antigens from crude mixtures. Antibodies may first be screened for use in IAC (Bonde, et.al., 1991). The selected antibodies are coupled to a chromatographic solid phase, e.g., cross-linked agarose, through covalent bonds, e.g., cyanogen bromide (CNBr) or other coupling chemistries targeting surface amino, hydroxyl, carboxyl or sulfhydryl groups of immunoglobulins to form a solid matrix. Recent coupling methods attempt site-directed immobilization of antibodies in an effort to optimize antigen-binding efficiencies, which are typically low, using classical coupling chemistries. One method of site-directed coupling is through carbohydrate groups of the F.sub.c immunoglobulin region to hydrazide-activated solid supports (e.g., Hoffman and O'Shannessy (1988)). The solid phase coupled to antibody is then packed in a chromatography column and equilibrated with buffer for binding to antigen. Mixtures of target protein and contaminants are equilibrated with binding buffer and then applied to the column. Non-adsorbed contaminants are removed by washout with various buffers. Elution occurs by use of chaotropic agents, extremes of pH, changes in ionic strength, etc. Elution is a critical aspect of IAC since the elution conditions may alter the biological activity of the immobilized antibody or the eluted antigen or both (reviewed by Jack, 1994).
IAC may be used to remove specific contaminants from a crude mixture. This mode was first applied to FSH purification using antibodies to hCG, which through cross reactivity to LH, effectively reduced LH contamination levels (Donini, et.al., 1966). Jack, et.al., (1987) and Great Britain patent number 8,510,177, utilized monoclonal antibody specific to FSH for IAC. The antibody was coupled to CNBr-activated Sepharose 4B. Samples were applied in 0.05M borate buffer, 0.5 M NaCl at pH 8.5 and non-adsorbed contaminants were eluted from the column with 0.05 M borate at pH 8.5. The bound FSH was eluted using 0.1M glycine, 0.5 M NaCl at pH 3.5. When using a sample containing the glycoprotein-enriched fraction from a side-fraction of human growth hormone obtained from frozen pituitaries, 47% of the applied FSH was recovered from the IAC procedure. The FSH was recovered at a specific immunological activity of 10,000 IU/mg (Biological specific activity=1.2.times. immunological). It contained 0.0014 IU LH per IU FSH and 0.93 .mu.IUs TSH/IU FSH (Jack, et.al., 1987). Another IAC method for FSH is described in U.S. Pat. No. 5,128,453. This method relies on a monoclonal antibody to FSH that is coupled to Sepharose 4B by divinylsulphone. The column and sample was equilibrated with 0.1M Tris, 0.3 M NaCl at pH 7.5 and the IAC procedure was performed at 4.degree. C. In this case, partially purified urinary FSH (hMG) was used as a sample. The sample was applied in the equilibration buffer and nonadsorbed materials were removed by continued elution with this buffer. Elution was accomplished by use of alkaline buffers, e.g., 1M ammonia and other eluent of pH&gt;11 and of ionic molarity higher than 0.8 M. The product of IAC was then subject to reverse phase HPLC on a C18 column to generate the final product. While the yield of FSH activity from the IAC step was not given, the final product had a specific biological activity of 6200 IU/Mg and had undetectable levels of LH contamination by RIA measurement. No other protein contaminants were detected by SDS-PAGE analysis. Other researchers have recently reported the use of dye affinity chromatography (DAC) in the purification of FSH from the bushtail possum. Their purification involved several steps including the use of Green A Matrix gel and Red A Matrix gel (Amicon, Inc.) for two sequential DAC procedures. The overall yield of their method was 12% (Moore, et.al., 1997).